Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

<p>Abstract</p> <p>Background</p> <p>Plasmid encoded ^<it>bla</it>CTX-M enzymes represent an important sub-group of class A β-lactamases causing the ESBL phenotype which is increasingly found in <it>Enterobacteriaceae </it>including <it>Klebsiella </it>spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.</p> <p>Methods</p> <p>Multiple displacement amplified DNA derived from 20 <it>K. pneumoniae </it>and 34 <it>K. oxytoca </it>clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.</p> <p>Results</p> <p>Nine out of 20 <it>K. pneumoniae </it>clinical isolates had a ^<it>bla</it>CTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 <it>K. oxytoca </it>clinical isolates. Molecular identification and differentiation between ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. <it>In silico </it>analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located ^<it>bla</it>OXY and K1-genes in <it>Klebsiella </it>spp. and K1-like genes in other <it>Enterobacteriaceae</it>.</p> <p>Conclusion</p> <p>The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of ^<it>bla</it>CTX-M, and ^<it>bla</it>OXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.</p

Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection

Up-Regulation of MUC2 and IL-1β Expression in Human Colonic Epithelial Cells by Shigella and Its Interaction with Mucins

BACKGROUND: The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis. METHODS: The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence. RESULTS: The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucin<guinea pig colonic mucin< Human colonic mucin. Invasion of Shigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells. CONCLUSIONS: Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells